Brain Organoids in Siberia

Great mini-brain capabilities

Alexandra Fedoseeva, "Science in Siberia"

If The Wizard of Oz had been written in our time, the Scarecrow could have gone straight to a research institute for brains, because they are already grown in laboratory conditions. There are only a few places in Russia where they work with new technology, one of them is in Novosibirsk Akademgorodok.

Of course, while these are not full-fledged, but mini-brains, or cerebral 3D organoids. "Here they live for about three months and grow to an average of 5 mm, while developing in the same way as the brain of a human embryo," says Tatiana A. Schneider, a junior researcher at the Institute of Cytology and Genetics SB RAS. The young scientist and her colleagues have been cultivating cerebral organoids for about a year in the department of molecular mechanisms of ontogenesis.

Of course, while these are not full-fledged, but mini-brains, or cerebral 3D organoids. "Here they live for about three months and grow to an average of 5 mm, while developing in the same way as the brain of a human embryo," says Tatiana A. Schneider, a junior researcher at the Institute of Cytology and Genetics SB RAS. The young scientist and her colleagues have been cultivating cerebral organoids for about a year in the department of molecular mechanisms of ontogenesis.

3D organoids are three-dimensional tissues that are very similar in structure to individual parts of real organs. Relatively speaking, these are the rudiments of organs grown from stem cells. The first organoids were obtained in Austria in 2013. Since then, the direction has been developing rapidly. Scientists have already created, for example, organoids of the kidneys, liver, lungs, and retina of the eye.

With cerebral organoids, experiments can be carried out that previously belonged to the realm of fiction. Mini-brains go through the same stages of development as the fetal brain, which means that they allow in vitro observation of the process of neurogenesis and how it is influenced by various factors. After all, the development of the fetus in the womb is a period when many hereditary diseases of the brain and nervous system are laid. Such experiments cannot be carried out with a real brain: since it is securely hidden in the skull, it is impossible to study it without harming the body, including the gray matter itself.

Mini-brains are used to study the formation of such severe mental disorders as schizophrenia and Alzheimer's disease. Also, these organoids have proved useful in studies of the mechanisms of action of the Zika virus, the pandemic of which occurred in 2014 (if the virus is carried by a pregnant woman, it causes microcephaly in the fetus). And with the help of cerebral organoids, they are trying to find the gene that makes us human. For example, scientists compared human organoids, orangutans and chimpanzees. There are works on "neanderthalization" of mini-brains: in the USA and in Sweden, in the group of biologist Svante Paabo, organoids with gene variants that differ in Neanderthals and homo sapiens were grown.

A pioneer in the field of growing brain organoids is Professor Madeline Lancaster from the University of Cambridge in the UK.

At the FITZ ICiG SB RAS, mini-brains began to be grown in order to study the effect on brain development of the CNTN6 gene encoding the protein contactin 6: in some people with mental retardation, the gene does not work. The researchers suggest that the result of this "breakdown" may manifest itself at the earliest, embryonic stage of cell life. If the experiments are confirmed, the data obtained can be used in prenatal screening – testing of potential health problems in the unborn child.

"We are, in fact, modeling the disease in the laboratory. They didn't come to this right away. At first, we tried to work with single-layer structures from grown neurons: this is a relatively simple procedure by today's standards, but it is not clear how such cells correspond to real neurons. Then human cells were transplanted into the brain of mouse embryos, but all this did not give the desired result," says the geneticist. The failures prompted Tatiana Schneider to try to get a three-dimensional cerebral organoid: several months were spent on selecting the right conditions for the growth of mini-brains, now the technology is working successfully.

The whole organoid

It all starts with the fact that a small sample of the upper layers of the skin is taken from a person. This is a painless procedure that is needed to get the so-called primary culture. To create organoids in the FIT ICiG SB RAS, skin cells of patients with mental retardation who have damage in the CNTN6 gene are used. Separate fibroblast cells are isolated from the connective tissue of the skin. They are transplanted into cups, where they grow and divide for a while. Then comes the stage of cell reprogramming: special viruses deliver proteins to the cell nuclei that trigger the process of converting fibroblasts into induced pluripotent stem cells (IPS cells).

Any type of cells can be obtained from induced pluripotent stem cells. These are complete analogues of embryonic stem cells, which are present in the embryos of all mammals at an early stage of development (at the blastocyst stage), and from which all organs and tissues are then formed.

To get a three-dimensional structure, scientists roll IPS cells into balls. After that, the cells begin to specialize, turning into three germ leaves, just like it happens in a real embryo: ectoderm, endoderm and mesoderm. In this case, scientists are interested in the ectoderm, from which neurons are formed, and then the nervous system, including the brain, is formed. "We are helping ectodermal cells. They gain an advantage over others in the form of certain components of culture media (nutrient solutions), therefore they begin to grow well. The rest either die or remain in very small numbers," says Tatiana Schneider.

In the early stages of brain development, cells must "understand" where it has "top" and where it has "bottom": this helps them migrate in the right direction and interact in the right way. To help the cells of an organoid determine its boundaries, scientists use a special gel. Tatiana Schneider explains: "The main component of the gel is proteins that concentrate on the surface of the organoids, the cells recognize them as a signal of the "upper boundary" (basement membrane), and inside the organoid itself, "inner boundaries" (apical membrane) are spontaneously formed."

After these manipulations, the containers with mini-brains are transferred to an orbital shaker. This is a platform that rotates in a circle at a certain speed in order for nutrients and oxygen to better penetrate into the organoid. The device is quite simple, but you need to choose the right rotation speed: if it is too large, the mini-brains may be damaged, too slow – the cells will not receive enough power.

Cups with mini brains
on an orbital shaker

 "I work with 10-12 lines of IPS cells at the same time. For each line there is a tablet with 96 holes, in each hole there is a future organoid. That is, there are only about a thousand potential mini-brains that need to be taken care of individually, to change the environment. The first two weeks are the most difficult, when they are especially sensitive. In order not to damage them, everything needs to be done very carefully," says Tatiana Schneider.

Many geneticists are trying to improve mini-brains with a variety of supplements. For example, they were squeezed from both sides with glass, and this force contributed to the formation of the similarity of brain convolutions in them. But the main problem of cerebral organoids is the lack of blood vessels. Because of this, their size and lifetime are limited: the cells located deep inside do not receive enough nutrients.

Can mini-brains think? According to Tatiana Schneider, this is unlikely: "We know little, or rather, practically nothing about the intrauterine thought process of a person. We don't have the tools to check whether organoids think. From what is available today, it is to measure the electrophysiological parameters of neurons. A preprint of an article by foreign scientists about the similarity of the encephalogram of mini-brains and premature babies has recently been published, but it is impossible to draw any conclusions from these results. Organoids have no organs of “expression of thoughts": they cannot write, draw or say. But most importantly, several parts of the brain are involved in the thinking process, and an organoid usually represents only one department."

In Novosibirsk Akademgorodok, organoids of the cortex (forebrain) are being studied, and in general, the hippocampus, cerebellum, and various nuclei have already been artificially grown in the world.  Most often these are individual organoids, since each type needs its own special conditions (that is, different chemical compounds).

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